Employing the pyruvate decarboxylase thiamine pyrophosphate (TPP), Mg(II) system we propose the following studies: 1. Models of TPP - amino acid binding (UV-VIS and fluorescence measurements); active-site labeling searching for important likely residues. 2. Synthesis of pyrimidine modified fluorescent TPP analogs to study binding kinetics and geometry. 3. Eu(III), Pm(III) as possible Mg(II) replacements to act as possible fluorescent and/or "nmr shift reagent" type reporters of kinetic, microenvironment (polarity) and binding geometry probes. 4. C13 and H1 nmr of coenzyme and substrate analog binding by chemical shift and T1 relaxation means. 5. C12/C13 kinetic isotope effects of enzymic and model decarboxylations to study rate limiting step on enzyme.